Priming of Citrullus lanatus var. Colocynthoides seeds in seaweed extract improved seed germination, plant growth and performance under salinity conditions

Citrullus lanatus var. Colocynthoide “Gurum” is an unconventional crop that can be utilized as a new source of edible oil and has the ability to grow in a variety of harsh conditions. To mitigate the adverse effects of salinity on seed germination and plant performance of C. lanatus, seeds were primed in the aqueous extracts of the seaweed Ulva lactuca before planting under greenhouse conditions. The aqueous extract of U. lactuca at 8% w/v led to maximal seed germination percentage and seedling growth of C. lanatus. Moreover, U. lactuca extract counteracted the negative effects of salt stress on the plant by significantly increasing the activity of SOD, CAT, and POD. The bioactive components of U. lactuca, e.g. glycine betaine and phenolic compounds can account for such beneficial role of algal extract on C. lanatus. Thus, priming of C. lanatus seeds in U. lactuca extract with various concentrations of U. lactuca extract can be employed as an effective practice for successful seed germination, improved plant growth and enhanced salt resistance, probably as a result of increased antioxidant enzymes activity and photosynthetic pigments.

Seaweed analysis. Assay of carbohydrates, lipids, proteins and glycine betaine. Soluble sugars were extracted from U. lactuca fronds according to the method adopted by Upmeyer and Koller 29 . To estimate insoluble sugars, the residue left after the extraction of soluble sugars was hydrolyzed by reflux in 0.2 N H 2 SO 4 in a boiling water bath for 1 h 30 . Carbohydrate fractions were determined by the anthrone method 31 . The lipid content of seaweed was estimated by extracting an aliquot of 3 g of the powdered fronds in petroleum ether for 6 h in the Soxhlet system according to AOAC 40 , and the extraction was continued using as a solvent 32 . The soluble protein content of the alga was determined according to the method of Lowry et al. 33 , while total protein content was calculated by multiplying the Kjeldahl nitrogen content 34 by 6.25. Assay of glycine betaine (GB) was carried out according to the method of Gorham 35 . Briefly, leaf extract was prepared by chopping 0.5 g of leaves in 5 mL of toluene-water mixture (0.05% toluene). The contents were shaken for 24 h at 25 °C. After filtration, 0.5 mL of the extract was mixed with 1 mL of 2 N HCl and 0.1 mL of potassium tri-iodide solution (containing 7.5 g iodine and 10 g potassium iodide in 100 mL of 1 N HCl) and the mixture was shaken in an ice-water bath for 90 min, and then 2 mL of ice-cooled water was added. After gentle shaking, 10 mL of dichloromethane (chilled at − 10 °C) was poured into the above mixture. By passing a continuous stream of air for 1-2 min, two layers were separated, the upper aqueous layer was discarded, and the absorbance of the organic layer was recorded at 365 nm. The concentration of GB was estimated by using a standard curve of GB in the range of 0-15 mg.
Proximate analysis of the seaweed. The detection of secondary metabolites (flavonoids, phenolics, alkaloids, saponins, tannins, and terpenoids) in U. lactuca aqueous extract was performed using the method of Sofowora 36 .
Quantitative determination of phenolic compounds. The phenolic content of U. latuca was determined using an Agilent-1100 HPLC system equipped with a quaternary gradient pump unit, an ultraviolet (UV) detector at 320 nm and a Zorbax Eclipse XDB-C18 analytical column (Agilent, USA) of 150 × 406 mm, 5 µm particle size. Elution was carried out at a flow rate of 0.075 mL min −1 at 23 °C. The mobile phase consisted of 8% acetonitrile, 22% isopropyl alcohol, and 70% formic acid solution (1%). All dissolved standards and samples were filtered through a 0.22 μm syringe filter before HPLC analysis. Seaweed aqueous extracts were frozen at − 20 °C for 24 h before drying in a frieze dryer, and the residues were dissolved in methanol (HPLC grade) before injecting into the HPLC; the injection volume was 20 μl. Identification of phenolic compounds was made by comparing the relative retention times of the sample peaks with those of the reference standards.
Preparation of U. lactuca seaweed aqueous extract. One hundred grams of U. lactuca powder were extracted by soaking in 1000 mL distilled water for 24 h, with shaking using an orbital shaker at 25 °C; the mixture was then centrifuged at 5000 rpm for 20 min to remove the debris. The extract was filtered through Whatman No. 4 filter paper, and the final volume was completed to 1 L with distilled water to obtain the stock extract (10% w/v). The resulting extract was stored at − 4 °C until used.
Effect of seaweed extracts on seed germination and plant growth of C. lanatus. The experiment was conducted during 2021/2022 at the Faculty of Science, Al-Azhar University (Girl Branch), Egypt. Plastic pots (20 cm in diameter and 20 cm in height) were filled with 3 kg of a clay/sand mixture (1:1) with the addition of the recommended doses of ammonium sulphate, ammonium nitrate and potassium sulphate fertilizers before sowing. The chemical analysis of the experimental soil mix was conducted at the beginning and the end of the experiment. Soil-water extracts were prepared by soaking 100 g of soil in 500 mL of distilled water  (12.16-15.25) for the pre-and post-cultivation soil samples, respectively; the water holding capacity was up to 13.0% 38 .
Twenty seeds of C. lanatus were sown in each pot and planted in April after soaking for 6 h in U. lactuca extracts at four concentrations (0, 3, 5, and 8%), The pots were watered with four salinity levels (0,100, 200, and 300 mM NaCl) every 7 days until the end of the experiment. After complete emergence (10 days), number of seedlings was counted 39 , and seedlings were sequentially thinned to five per pot across the following five days. Pots were arranged in a randomized complete block design with three replicates in the greenhouse at 34 °C on average with 16 h of light and 8 h of darkness.
Plant harvesting and analysis. Assay of plant growth and photosynthetic pigments. Three representative plant samples were taken from each treatment for measuring of growth traits after 45 days of growth: shoot length (cm), root length (cm), and the number of branches were measured; leaf area (cm2), was measured using leaf area meter (SYSTRONICS Leaf Area Meter-211). Photosynthetic pigments (chlorophyll a [Chl a], chlorophyll b [Chl b] and carotenoids [C(x + c)] were quantitatively determined spectrophotometrically according to the procedure adopted by Metzner et al. 40 and, Abbas 41 . An aliquot of the fresh leaf tissue (0.5 g) was homogenized in a mortar with 10 mL of 80% acetone; the homogenate was centrifuged at 3000 rpm for 15 min at room temperature, and the supernatant was stored at 4 °C. The absorbance of the extract was measured at 645, 663 and 480 nm using a spectrophotometer (VEB Carl Zeiss). Chlorophyll a, chlorophyll b, and carotenoids were determined as µg g −1 leaf fresh weight using the following equations: The concentrations of chlorophylls and carotenoids were expressed as mg g −1 fresh weight (FW) of plant material.
Estimation of C. lanatus antioxidant enzyme activities. An aliquot of the fresh leaves (0.5 g) was homogenized in liquid nitrogen with the addition of 5 mL of 0.2 mol L −1 of sodium phosphate buffer solution (pH 7.8). Homogenates were centrifuged for 20 min at 4 °C, and supernatants were immediately used to determine enzyme activity. Total soluble protein content of the leaves was determined according to the procedure of Bradford and Williams 42 . Superoxide dismutase (SOD) activity was assayed in terms of the extent of inhibition of the photochemical reduction of p-nitro blue tetrazolium chloride (NBT) 43 . Catalase (CAT) activity was determined based on the rate of disappearance of H 2 O 2 , as measured by the decline in absorbance at 240 nm 44 . Peroxidase activity (POD) was calculated from the rate of formation of guaiacol dehydrogenation product and was expressed as mmol GDHP min −1 mg −1 protein 45 . Data analysis. The data were subjected to two-way ANOVA according to Snedecor and Cochran 46 using SPSS ver. 26.0, and considering p < 0.05 as a significant level. Tukey's HSD is used to test differences among sample means for significance. Spearman correlation was done by Sigmaplot 12.5, to assess the effects of seaweed and salinity on germination, seedling growth and plant performance.
Ethical approval. All the steps of experimentation on C. lanatus var. Colocynthoide plants, including the collection of plant material, are in compliance with relevant Institutional, National, and International guidelines. The greenhouse studies were conducted in accordance with local legislation and with permissions from our institutes and complied with the IUCN Policy Statement.

Results and discussions
Chemical composition of Ulva lactuca. Results of the biochemical analyses of U. lactuca fronds indicate that the algal biomass contained (one dry weight basis): 0.93 mg g −1 total carbohydrates, 3.01 mg g −1 of lipids, and 3.21 mg g −1 protein, in addition to appreciable content of glycine betaine (5.13 mg g −1 ) and total phenolics (0.0188 mg g −1 ). This result is in agreement with Latique et al. 47 who found that Ulva rigida extract is rich in soluble sugars, polyphenols, and proteins which might be necessary for the stimulation of antioxidant enzymes. Glycine betaine is an osmoregulatory molecule that enables plant cells to cope with salt stress 48 .
The aqueous extract of U. lactuca contained appreciable content of saponins, alkaloids, and tannins along with high content of flavonoids and phenolics but lacks terpenes. Hence, the high phenolic and flavonoid content of U. lactuca extract may account for its high efficiency in the enhancement of C. lanatus and alleviation of the impact of salt stress.
Nine free compounds were identified in the aqueous extract of U. lactuca extract (Table 1) by reverse-phase high-performance liquid chromatography (HPLC). These compounds were identified based on their relative retention times. Ascorbic acid and coumaric acid amounted to about 48.59 and 36.23 µg g −1 DW, respectively, and were the two main components in U. lactuca extract. Caffeic, ferulic, protocatechuic and pyrogallic acids, and resorcinol all recorded moderate amounts of 29 Effect of U. lactuca an aqueous extract on C. lanatus seed germination. Figure 1 shows that priming of C. lanatus seeds in U. lactuca extracts significantly improved germination of seeds, and the increase was most evident at high salinity, Increasing concentration of algal extract during priming from 0 to 0.8% increased.
Germination percentage of C. lanatus seeds by 24.5%, 53.9%, 61.3%and 66.7% at 0.0, 100, 200 and 300 mM NaCl salinity, respectively. Increasing NaCl salinity reduced seed germination and the magnitude of reduction differed according to the concentration of the priming algal extract. Whereas increasing salinity from 0 to 300 mM NaCl led to progressive 20.3% reduction in seed germination in absence of algal extract, the reductions were relatively mild and amounted to 80.3%, 65.0%, 60.0 and 60.0% at 3%, 5% and 8% algal extract, respectively. The germination in C. lanatus was affected significantly by algal concentrations (F = 44.73, p < 0.000) and salt concentrations (F = 14.07, p < 0.000), while, there was interaction within algal concentrations and salt concentrations (F = 6.25, p < 0.02) ( Table 2). Salt stress presents a threat to C. lanatus germination, growth parameters, and photo/biosynthetic materials. Therefore, Algal extracts from U. lactuca can be used as priming practices to promote C. lanatus cultivation under salinity stress. Seeds are usually soaked in an osmotic solution that allows them to imbibe water and go through the first stages of germination 54 . Seed pretreatment with algal extract that contains bioactive substances can stimulate the metabolic process in C. lanatus seeds under the impact of salinity stress. Seed presoaking can stimulated the antioxidant enzyme activities that play a vital role in C. lanatus resistance against salt stress. The beneficial et of algal extract in improvement of seed germination percentage and the subsequent plant growth of C. lanatus under the impact of salinity can be attributed to its effect on seed surface permeability to salts, and its content of bioactive components in U. lactuca extract of glycine betaine and phenolics and ascorbic acid could potentially participate in the alleviation of salinity stress. Thus, priming of C. lanatus seeds with U. lactuca extract could induce many physiological and biochemical changes that can stimulate seeds to germinate faster and increase the germination percentage of C. lanatus. Algal extracts increase seed germination and seedling growth in several plants spices with alleviation of the impact of a biotic stress [55][56][57][58] and fruit production 59 . The priming of C. lanatus seeds in U. lactuca aqueous extract improved plant growth and photosynthetic pigments significantly under the impact of salinity. Shoot length, root length and leaf area as well as the content of photosynthetic pigments have been reported to increase in the salinized plants in response to presoaking of seeds in Ulva fasciata extract 60,61 . The beneficial effect of primed cherry tomato seeds in Ulva flexuosa extract on could be attributed to the high concentration of glycine betaine in the algal extract 62 . In support to this claim, exogenous application of glycine betaine significantly increased plant height and the number of leaves of Dalbergia odorifera seedlings under mild and severe salinity 63 .

Effect of U. lactuca extracts on photosynthetic pigments of C. lanatus. The effects of salinity and
algal priming on leaf photosynthetic pigments of C. lanatus were substantial (Table 3), with relatively stronger effect of salinity than algal priming and very highly significant interaction, except for the non-significant interaction on carotenoid content. The content of chlorophyll a, chlorophyll b, and carotenoids increased progressively by increasing the concentration of U. lactuca extract. The increase in chlorophyll a, chlorophyll b, and carotenoids at 8% aqueous algal extract amounted to 469%, 918%, and 961% respectively above the non-treated control (Fig. 3). Our results are in agreement with Ahmed et al. 64 who found that the content of chlorophylls a and b and carotenoids in cotton leaves was significantly improved by algal extract as compared to the control. The application of U. rigida extract to wheat plants under salt stress conditions led to a significant increase in Total  Control +100 NaCl UL 3% +100 NaCl UL 5% +100 NaCl UL 8% +100 NaCl Control +200 NaCl UL 3% +200 NaCl UL 5% +200 NaCl UL 8% +200 NaCl Control +300 NaCl UL 3% +300 NaCl UL 5% +300 NaCl UL 8% +300 NaCl Control (DW) UL 3% + No salt UL 5% +No salt UL 8% + No salt Control +100 NaCl UL 3% +100 NaCl UL 5% +100 NaCl UL 8% +100 NaCl Control +200 NaCl UL 3% +200 NaCl UL 5% +200 NaCl UL 8% +200 NaCl Control +300 NaCl UL 3% +300 NaCl UL 5% +300 NaCl UL 8% +300 NaCl UL 3% + No salt UL 5% +No salt UL 8% + No salt Control +100 NaCl UL 3% +100 NaCl UL 5% +100 NaCl UL 8% +100 NaCl Control +200 NaCl UL 3% +200 NaCl UL 5% +200 NaCl UL 8% +200 NaCl Control +300 NaCl UL 3% +300 NaCl UL 5% +300 NaCl UL 8% +300 NaCl UL 3% +100 NaCl UL 5% +100 NaCl UL 8% +100 NaCl Control +200… UL 3% +200 NaCl UL 5% +200 NaCl UL 8% +200 NaCl Control +300… UL 3% +300 NaCl UL 5% +300 NaCl UL 8% +300 NaCl Leaf area (cm2)  www.nature.com/scientificreports/ Chl (T-Chl), Chl-a, and Chl-b 65,78 . Leaf pigments are a major physiological attribute that is directly related to the photosynthesis process under various environmental conditions 66 . This increase in chlorophyll content could be a result of the protection of chlorophyll from degradation that might be attributable to betaines of the seaweed extract 67 . Seaweed extracts by virtue of their high mineral content, particularly, magnesium and can increase leaf chlorophyll and carotenoid concentration 68  www.nature.com/scientificreports/ enhance the activity of SOD, CAT, and POD in C. lanatus along with improving salt tolerance that boosts the plant development under stress conditions. Salinity tolerance is strongly associated with enhanced antioxidant enzyme activities as well as high content of non-enzymatic antioxidants 73 . The role of antioxidant enzymes in salt-resistance has been reported in many plant species (Arabidopsis, barley, pea, and tobacco) [74][75][76] . Macroalgae have been recognized as a rich source of antioxidants 77 . Algal extract of U. rigida antagonizes the harmful effects of abiotic stress by increasing the activity of antioxidant enzymes 78 . Ulva rigida extract led to a significant promotion in antioxidant enzyme activity in salt-stressed wheat plants 79 . Superoxide dismutase (SOD) is the first enzyme that acts against reactive oxygen species 80 . A significant enhancement in catalase (CAT) and superoxide dismutase (SOD) activities was observed in wheat plants treated with 25% of U. rigida extract under salt stress conditions 81 . Superoxide dismutase, catalase and peroxidases as well as ascorbate peroxidase have been claimed to protect plants against the harmful effects of reactive oxygen species 82,83 . The content of soluble sugars, polyphenols, and proteins in Fucus spiralis extract may account for the increased salt resistance of durum wheat, probably via stimulation of antioxidant enzymes 47 . Furthermore, germination and growth traits were positively correlated to enzyme activity and pigments (Table 4)  www.nature.com/scientificreports/ membrane mending 84 . The promoting priming effects of algal extracts in plant growth can contributed to the supply of plant nutrients from the soils 85 . Some of these elements are directly related to leaf pigments or catalyze some physiological process that leads to promoting the production of biochemical substitutes 86 . Seaweed extracts enhanced the biochemical constituents in crops 87 . Algal extracts can enforce different physiological and biochemical mechanisms to improve the salinity tolerance of plants 60 . Seaweed extract of U. rigida can increase salt stress tolerance and protect wheat plants against oxidative deterioration 88 . Macroalgae could enhance plant salt tolerance through the increasing antioxidant compounds, compounds such as ascorbic acid and polyphenols, which might be necessary for the stimulation of antioxidant enzymes 47 . Algal extracts can relieve the effects of salt stress through the activation of metabolic pathways that contribute to promoting plant growth and yield 89 . The enhancement of plant salt resistance was associated with an increase in water use efficiency, photosynthetic activity, phenolic compounds, and ROS scavenging activity. Glycine betaine is involved in mitigating salt stress in plants through osmotic adjustment and ROS scavenging 90 . Ulva rigida extract is rich in glycine betaine, which delays the loss of photosynthetic activity by inhibiting chlorophyll degradation 67 . Exogenous application of glycine betaine significantly increased plant height and number of leaves of Dalbergia odorifera under mild and severe salinity stress conditions 63 . It could be concluded that the aqueous extract of the seaweed U. lactuca can boost seed germination percentage, plant growth, photosynthetic pigments and performance of C. lanatus under salt stress conditions by virtue of their contents of bioactive components and biochemical parameters, they could stimulate the tolerance response of C. lanatus toward biotic stress of salts. Therefore, this research advised breeders to use seaweed extract as an effective seed priming technique to promote C. lanatus germination and growth is an effective under salt stress conditions for increasing plant productivity.